Initial evaluation of a convection counter streaming galvanization technique of sex separation of human spermatozoa*

Transcription

1 FERTILITY AND STERILITY Copyright " 1982 The American Fertility Society Printed in U.SA. Initial evaluation of a convection counter streaming galvanization technique of sex separation of human spermatozoa* James F. Daniell, M.D.t Carl M. Herbert, M.D. John Repp, A.M.T. Charles A. Torbit, Ph.D. Anne Colston Wentz, M.D. Center for Fertility and Reproductive Research, Department of Obstetrics and Gynecology, Vanderbilt University Medical School, Nashville, Tennessee A new method for separating X and Y human spermatozoa called convection counter streaming galvanization " was evaluated. The method was independently performed by this semenology laboratory with the use of the special separation equipment and extending media provided by its developer, Dr. Bhairab C. Bhattacharya. The mean number of Y spermatozoa increased from 48% to 77% in the separated fraction predicted to be Y-enriched. The fraction predicted to be X-enriched increased from a mean of 52% to 77%. The one separation process allowed accumulation of both enriched fractions simultaneously. The separated portions of spermatozoa maintained good motility and penetration of cervical mucus but produced a mean recovery concentration in the X-and Y -enriched fractions of only 15% to 16% of the preseparation concentration. Fertil Steril38:233, 1982 There have been many attempts to separate X and Y chromatin-bearing spermatozoa in order to influence the sex of the offspring. Reported experimental methods include millipore filtration,i,2 albumin gradient sedimentation,3 centrifugation, 4 electrophoretic separation,5 and" Sephadex gel filtration. 6 Dr. B. C.Bhattacharya has developed a new process that reportedly allows recovery of both X and Y spermatozoa using a special particle-free semen extender and a special apparatus for separation. His initial reports about this technique, Received March 16, 1982; revised and accepted May 14, *Presented at the Thirty-Eighth Annual Meeting of The American Fertility Society, Marcil 20 to 24,1982, Las Vegas, Nevada. treprint requests: James F. Daniell, M.D., Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Vanderbilt University Medical School, Nashville, Tennessee which he has called "convection counter streaming galvanization," seem promising.7, 8 In an attempt to evaluate this technique's potential for human use, Dr. Bhattacharya's apparatus, extender medium, and specific techniques, which he describes in a book,9 were utilized in our semenology laboratory for the separation of donor human spermatozoa. This study reports our initial experience in humans with this separation process, as measured by Y -body fluorescence microscopy. The effect of the separation process on sperm motility, viability, and density have been noted, as well as the ability of the separated spermatozoa to penetrate cervical mucus (CM) in capillary tube tests. MATERIALS AND METHODS Fresh" semen samples were obtained by masturbation after 2 days of abstinence in 33 men from our pool of sperm donors. A routine semen analysis was performed. Using the universal se- Daniell etal. Sex separation of human spermatozoa 233

2 Figure 1 The prototype apparatus for performing the convection counter streaming galvanization technique for separation of human spermatozoa. men extender medium (UEM) developed by Dr. Bhattacharya and the convection streaming, galvanic cell apparatus (Fig. 1) provided for this study by Applied Genetics Limited (New Brunswick, NJ), our chief semenology technician (J. R.) performed the separation after he had been personally instructed in the procedure by Dr. Bhattacharya in our laboratory. The extender medium used has been developed and patented (U.S. Pat. Number ) by Dr. B. C. Bhattacharya. The medium contains longrange buffering agents like glycine, alanine, a colloidal agent, lecithin, and other biologically active peptides from egg yolk and antibiotics as antibacterial agents but no lysing agent such as lysozyme and lysolecithin of egg yolk. The medium contains no particle over 0.2!-Lm in size; hence it is' sterile. The isotonicity and good buffering capacity in the neutr~l region make the medium suitable for semen of all species. The total fresh donor semen specimen was incubated at 37 C for 15 minutes, and the volume was recorded. A beaker containing 40 ml of UEM was simultaneously incubated at 37 C, and after Daniell et ai. Sex separation of human spermatozoa minutes 10 ml of UEM was added to the entire semen specimen with gentle agitation to ensure uniform distribution. A 10-.u sample was then taken for analysis of sperm concentration (corrected for the addition of 10 ml UEM), motility, and viability. After 45 minutes offurther incubation at 37 C, the diluted specimen was mixed with the remaining 30 ml of UEM. Next the specimen was placed in a cooling room that contained the separation apparatus and cooled to a temperature of 5 C over 1 hour. Simultaneously, an additional 200 ml of UEM was also separately placed in the cool room for 1 hour. After the cooling period, the additional UEM was used to prime the separation apparatus and aid in transfer of the specimen. The separation process was run for 1 hour at 5 C. After completion of the process, the various samples were collected in individual centrifuge tubes labeled "male specimen" (taken from the anode), "female specimen" (taken from the cathode), and "residual specimen" (unseparatedpool); These specimens were centrifuged for 15 minutes at 600 x g. The supernatants were decanted to a volume of 1 ml, and with gentle agitation the specimens were resuspended for uniform distribution in the residual universal extender. After 15 minutes of equilibration at room temperature, small aliquots were taken from the male and female specimen tubes and examined for sperm concentration, motility, and viability. In 13 studies an additional 'aliquot was taken from all three specimen tubes, warmed to 37 C, and used in sperm-cervical mucus penetration tests (CMPTs),lO using known normal CM. In preparation for determining X:Y sperm distribution, all three samples were washed four to six times in Tyrode's solution. A drop from each specimen was then carefully smeared on a microscope slide, allowed to dry, and fixed for 20 minutes in a 3:1 methanol-acetic acid solution. Two separate sets of slides of each enriched fraction were labeled and stored after fixation for future independent determination of X: Y distribution. To determine the Y-body-positive sperm content of the specimens, the slides were immersed in a 5 mg% solution of quinacrine mustard for 20 minutes, rinsed in distilled water, and covered with several drops of acid buffer* ~nd a coverslip. The slides were immediately scanned under a *Acid buffer: 0.25 M Na2HPo4.2H2, Part A; 1 M citric acid, Part B; 3 parts A to 1 part B; adjust ph to 5.6 with Hel.. Fertility and Sterility

3 Table 1. Effect of Separation on Motility and Count of Spermatozoa (Bhattacharya Technique) Volume Prior to separation controls Male-enriched fraction (anode) After separation Female-enriched fraction (cathode) Numbe'i{.ml Percentage Percentage Number/ml Percentage Percentage Number/ml Percentage Percentage (x 10} motile live (x 1~ motile live (x 1~ motile live ml Mean SD" Number "Standard deviation ' , '., I! I, i '1, Zeiss fluorescence microscope (Carl Zeiss, Inc., New York, NY), the fluorescent Y-bodies were noted, and the percentages were recorded after 200 spermatozoa were counted.ll All counts were made by one author (J. R.), who has had extensive previous experience viewing fluorescent Y -bodies in human spermatozoa. RESULTS Table 1 shows the pre- and postseparation findings on the semen specimens. Prior to separation, the donor semen specimens demonstrated normal volumes, sperm concentration, and motility and viability percentages. After separation, the motility and viability percentages were unchanged. The volume collected at each pole was constant at 1 ml. The concentration of sperm after separation was considerably reduced. Table 2 reveals the change in sperm concentration at each pole of the apparatus after separation. Expressed as a percentage of the original preseparation concentration at both cathode and anode, after separation the recovery was approximately 15% of the original concentration. Table 3 reveals a normal X: Y sperm distribution prior to separation. After separation the fractions collected at the anode contained an average of 77% Y-fluorescing spermatozoa with a range of 68% to 92%. Similarly, the fraction collected froin the cathode demonstrated an average of 76% non Y-fluorescing spermatozoa with a range of 67% to 88%. The results of in vitro CMPTs performed on pre- and postseparation sperm samples are shown in Table 4. There was no significant change in sperm-penetrating capacity subsequent to passage through the apparatus. DISCUSSION Throughout recorded history, there has been much importance given to the sex and sequence of progeny for inheritance and other purposes. Thus, for centuries potential patients and interested health care providers have attempted to influence the sex of offspring. These attempts can be divided into three different categories of action. First, there have been attempts to time coitus or otherwise influence the number of male or female spermatozoa entering the upper reproductive tract by manipulations prior to or during coitus. There have been reports of success using various manipulations,12 but none of these proposed suggestions has been adequately scientifically evaluated. For proper perspective it must be remembered, as Dmowski et al. state, "All methods, including folk recommendations and ancient techniques, can claim at least 50% success.,,13 A second alternate, selective abortion following, genetic amniocentesis, has been employed for the purpose of avoiding the passing on of certain hereditary diseases. However, this method is expensive, often stressful on both patient and physician, and carries certain risks associated with midtrimester abortion. Table 2. Recovery of Spermatozoa Following Separation by the Bhattacharya Technique Mean SD b Percen~eof concentration reo covered at anode g PercentBlre of concentration recovered at,cathode "# spenn/cc in specimen # spenn/cc in original specimen x 100 = percentage of concentration recovered. bstandard deviation. Daniell et ai. Sex separation of human spermatozoa 235

4 Table 3. Results of Sex Separation Procedure on Y -Body Fluorescence of SperTYULtozoa Before and After Separation (33 Separations) Mean SDc ay -bearing spermatozoa. bx-bearing spermatozoa. CStandard deviation. Prior to separation Male-enriched fraction (anode) Female-enriched fraction (cathode) Finally, as reported in this paper, there have been attempts at separation of semen obtained by masturbation in fractions of X or Y spermatozoa. Subsequently, artificial insemination or in vitro fertilization could be performed with that portion of semen containing the type of spermatozoa desired. The couple who wishes to attempt a more scientific preselection of the sex of their child, for whatever reason, and who can accept artificial or in vitro insemination, may desire in vitro sperm separation techniques if they are available. Of all the reported preselection techniques, the one most widely available and used is that reported by Ericsson et al. 3 This technique, which is clinically available in several centers, only allows concentration of male spermatozoa and thus is of no help to the couple who desire a female child or carry an X-linked genetic disorder. The fact that the live births following the Ericsson technique for insemination demonstrate birth ratios predicted by Y-body staining and occur without apparent decreased fertility, increased abortion rates, or abnormal births is encouraging,13 and suggests that other techniques of sperm separation that result in a high separation of motile spermatozoa could result in similar birth ratios with normal offspring. A recent report by Quinlivan et al. 14 indicated that one laboratory using two different techniques for separation (Ericsson et al. 3 for male-enriched and Steeno et al. 6 for female-enriched spermatozoa) may be able to concentrate X or Y spermatozoa with efficiency approaching 75%. This study is the first independent evaluation of a single technique of sperm separation that may hold promise for selection of either a male or a female child with greater than 75% potential for success. The separation provides simultaneous specimens of high male sperm concentration and high female sperm concentration, thus potentially allowing selection of either sex by a single process. The technique requires a special apparatus Table 4. Results of in Vitro Sperm Penetration Tests with Sex-Separated SperTYULtozoa a Prior to separation After separation Female-en riched frac- tion Male-enriched fraction and special extending media, but the procedure is technically simple and requires only 3 hours when performed by a trained technician. The decrease in sperm concentration in the separated fractions is similar to the decrease seen with the successfully employed technique of Ericsson et al. 3 The low sperm recovery after separation may decrease conception rates in certain settings, such as moderate oligospermia, or even in normal couples desiring sex preselection. However, since the spermatozoa in these separated fractions maintain a high degree of motility and penetrate CM adequately, it would appear that pregnancy rates with spermatozoa separated by this technique would be satisfactory for certain clinical applications. This is the subjective impression of the only physician currently doing clinical inseminations for sex selection using the Bhattacharya technique.15 In conclusion, fractions of highly motile spermatozoa with good CM penetrating capacity can be separated by the Bhattacharya technique. Y body fluorescence of these fractions indicates definite alteration of the sex ratio. However, separation of the X and Y spermatozoa is not complete, and the ratio, although increased, is not as high as is suggested to be possible by Bhattacharya et al. 8 This preliminary report suggests that the Bhattacharya technique of sperm separation may offer hope of an altered sex ratio for couples desiring either a male or a female child. Further inde- Mean penetration of mm mm mm spermatozoa for 13 specimens mm mm mm aone hour in 50 mm capillary tubes. 236 Daniell et ai. Sex separation of hutyuln spertyultozoa Fertility and Sterility

5 pendent evaluations of this process and carefully controlled clinical trials involving insemination of patients with enriched fractions obtained by this technique will be necessary for us to determine whether significantly altered birth ratios can be achieved without reduced pregnancy rates or the occurrence of abnormal births. REFERENCES 1. Broer KH, Dauber U, Kaiser R, Schumacher GFB: The failure to separate human X- and Y-spermatozoa by the millipore filtration technique. J Reprod Med 20:67, Adimoelja A, Hariadi R, Amitaba 1GB, Adisetya P, Soeharno: The separation of X- and Y-spermatozoa with regard to the possible clinical application by means of artificial insemination. Andrologia 9:289, Ericsson RJ, Langevin CN, Nishino M: Isolation of fractions rich in human Y sperm. Nature 246:421, Shilling E, Thormaehlen D: Enrichment of human X and Y spermatozoa by density gradient centrifugation. Andrologia 9:106, Shishito S, Shirai M, Sasaki K: Galvanic separation of X and Y-bearing human spermatozoa. Int J Fertil 20:13, Steeno 0, Adimoelja A, Steeno J: Separation of X and Y bearing human spermatozoa with the Sephadex gel-filtration method. Andrologia 7:95, Bhattacharya BC, Shome P, Gunther AH: Successful separation of X and Y spermatozoa in human and bull semen. Int J Fertil 22:30, Bhattacharya BC, Evans BM, Shome P: Semen separation technique monitored with greater accuracy by B body test. Int J Fertil 24:256, Bhattacharya BC: The counter current galvanization technique of sex separation. In Guide to Pre-arranging the Sex of Offspring. Ralston, Nebraska, Action Press, 1977, p Kremer J: A simple penetration test. Int J Fertil1O:29, Pearson P: The use of new staining techniques for human chromosome identification. J Med Genet 9:264, Vear CS: Preselection determination. Med J Aust 2:700, Dmowski WP, Gaynor L, Rao R, Lawrence M, Scommegna A: Use of albumin gradients for X and Y sperm separation and clinical experience with male sex preselection. Fertil Steril 31:52, Quinlivan WLG, Preciado K, Long TL, Sullivan H: Separation of human X and Y spermatozoa by albumin gradients and Sephadex chromatography. Fertil Steri137:104, Personal communication with Dr. Ray Schulte, Omaha, Nebraska Daniell et ai. Sex separation of human spermatozoa 237